Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates ( R 2 * and R 2 ) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R 2 ′ ( R 2 * ‒ R 2 ) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like m agA . Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

Magnetic resonance imaging can be used to track cellular activities in the body using iron-based contrast agents. However, multiple intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast: a need to differentiate among those mechanisms exists. In hepcidin-mediated inflammation, for example, downregulation of iron export in monocytes and macrophages involves post-translational degradation of ferroportin. We examined the influence of hepcidin endocrine activity on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron import and export activities, similar to alternatively-activated macrophages. Iron import and export were examined in cultured P19 cells in the presence and absence of iron-supplemented medium, respectively. Western blots indicated the levels of transferrin receptor, ferroportin and ubiquitin in the presence and absence of extracellular hepcidin. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated to transverse relaxation rates at 3 Tesla using a gelatin phantom. Under varying conditions of iron supplementation, the level of ferroportin in P19 cells responds to hepcidin regulation, consistent with degradation through a ubiquitin-mediated pathway. This response of P19 cells to hepcidin is similar to that of classicallyactivated macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was different in hepcidin-treated and untreated P19 cells: slope, Pearson correlation coefficient and relaxation rate were all affected. These findings may provide a tool to non-invasively distinguish changes in endogenous iron contrast arising from hepcidin-ferroportin interactions, with potential utility in monitoring of different macrophage phenotypes involved in pro-and antiinflammatory signaling. In addition, this work demonstrates that transverse relaxivity is not only influenced by the amount of cellular iron but also by its metabolism.MRI is a non-invasive imaging method that can be used to track cellular activities involved in different diseases. Toward achieving molecular imaging capability, various iron-based exogenous and endogenous contrast agents have been developed to enhance image contrast and improve molecular imaging 12,13 . In addition, cellular iron metabolism might also be expected to influence the accumulation of contrast agents and their detection by MRI 14 . In the case of iron-exporting cells (particularly pro-and anti-inflammatory macrophages), little is known about how their distinct iron regulation may be distinguished by MRI. To investigate this, we used the multi-potent P19 stem cell model with high iron import and export activities 15 , the latter of which corresponds with high FPN 14 . In this regard, P19 cells resemble macrophages 5 and are a convenient model of iron regulation related to inflammation signaling. We examined the effect of varying extracellular iron supplementation and hepcidin on MR cont...